Abstract

Within this work, time-lapse microscopy and quantitative analysis of motion parameters were used to characterize Ras regulation of the phenomena determining directional cell migration. The behaviour of NIH3T3 and NIHRasV12 fibroblasts, expressing RasV12, a constitutively activated variant, was characterized in terms of kinetic, morphological and biochemical features.
Constitutive RasV12 activation results in both morphological changes and increased proliferation and speed, and confers to NIH3T3 cells the ability to close the wound faster in wound healing assays. Since neither increased cell proliferation nor higher speed explain, alone, the accelerated wound closure, other parameters, descriptive of specific aspects of cell motion, such as linearity, persistence and directionality, were quantitatively evaluated. For both NIH3T3 and NIHRasV12 fibroblasts, the wound acts as a directional stimulus, but the presence of constitutively activated RasV12 amplifies the directional response: NIHRasV12 migration is characterized by paths more directional and directed towards the wound. The role of the effectors through which Ras drives directional cell migration, was evaluated by separately blocking two main signaling pathways downstream from Ras, PI3K and MAPK pathway. The inhibition of PI3K pathway reduced speed but not the directional component of cell migration; instead, the selective block of ERK activation showed that directionality, more than speed, is associated with MAPK pathway and strictly depend on ERK phosphorylation.
Immunofluorescence stainings showed that ERK activates at the wound edge immediately after wound. The biochemical analysis, by western blot, of cell extracts enriched with cells located in the proximity of a wound, showed that ERK phosphorylation occurs in a prolonged and biphasic way. Moreover, both peaks of ERK activation resulted dependent on neo-phosphorylation by MEK.
Actin modifications, induced by the wound, are impaired by the inhibitor of ERK activation, in both NIH3T3 and NIHRasV12 fibroblasts, suggesting that the cellular response, responsible for starting and driving directional movement, depend on early ERK activation by Ras, after the wound stimulus.